Hepatic, microsomal, cytochrome P450 monooxygenases provide a crucial protective role in the metabolism and clearance of a wide array of drugs, toxins and endobiotics. The long term objective of this application is to identify structural features that determine the substrate selectivity of individual human P450 enzymes. Active site characteristics affect substrate selectivity, inhibitory potential, the regioselectivity of drug oxidation, as well as the occurrence of drug-drug interactions and activation kinetics. Comparison of recent structures for P450s 1A2, 2A6, 2C8, 2C9 and 3A4 in complex with substrates or inhibitors indicate unique aspects of each active site that contribute to substrate selectivity including the hydration state, the chemical characteristics and positioning of active site residues, as well as the changes imposed on these binding determinants by alterations in active site topology. This underscores the necessity to determine multiple structures of individual enzymes in complex with structurally dissimilar drugs to understand the contribution of conformational flexibility to drug binding. Specific changes in the active site architectures of P450s 1A2, 2D6, 2C19 and 2C9*3 induced by interaction with chemically diverse substrates, inhibitors and activators, alone or in combination, will be identified to delineate the range of adaptive changes that can occur for each enzyme. P450 2A6 is the principal nicotine oxidase in humans. Analysis of the 2A6 active site in complex with different inhibitors indicates several relatively static features that can be productively exploited for the structure based design of more efficacious and selective inhibitors that would have potential benefits as therapeutic aids for smoking cessation. Lead compounds will be assessed for complementarity with the active site, strength of binding, and specificity for 2A6 relative to other P450s. The active site topologies of P450s 1B1, 3A5 and 3A7 will also be determined to identify structural characteristics that control the important functional contribution made by these enzymes to drug metabolism. Collectively, these studies will address significant gaps in our knowledge of P450 structure as it relates to function, and provide important information and tools for prediction of drug metabolism.